This R21/R33 proposal combines the efforts of an international team to develop tools and methods to monitor a subset of the ganglioside metabolome in single mammalian cells. In the two-year duration R21 phase of the project, a set of eight fluorescent substrates and standards will be synthesized using chemical and enzymatic methods. The set consists of all known metabolites along two different glycolipid metabolic pathways. The standards and substrates will be prepared with four different fluorescent labels, so that different points in the metabolic pathway can be accessed simultaneously. NG108-15 and/or PC12 cells will be incubated with the substrates, which will undergo biosynthesis and biodegradation. Only those reaction products that preserve the fluorescent label will generate a detectable signal in our instrument, allowing careful dissection of specific metabolic pathways. A four-spectral channel laser-induced fluorescence detector will be assembled for this project. This detection system will allow the subattomole detection of four different sets of metabolic products, which will provide a particularly powerful tool for the characterization of metabolites along different pathways. During the one-year duration R33 phase of the project, this set of reagents and instrumentation will be applied to the study of metabolism in single cells. The long-term goal of this project is to monitor the complete ganglioside metabolome in single neurons.